Inhibition of reactive oxygen species and protection of mammalian cells

ABSTRACT

Methods and compositions useful for neuronal protection in retinal cells in vitro and the protection of mammalian cells from reactive oxygen species in vivo are provided. Ultrafine nano-size cerium oxide particles, less than 10 nanometers in diameter, have been provided to decrease reactive oxygen species (ROS) in retina tissue that generates large amounts of ROS. These reactive oxygen species (ROS) are involved in light-induced retina degeneration and age-related macular degeneration (AMD). Cerium oxide nanoparticles have been used to promote the lifespan of retinal neurons and protect the neurons from apoptosis induced by hydrogen peroxide in vitro and in vivo. The neuronal protection in retinal cells is achieved by decreasing generation of intracellular reactive oxygen species (ROS). Thus, cerium oxide particles are used to promote the longevity of retinal neurons in vitro and mammalian cells in vivo.

This invention claims the benefit of priority from U.S. ProvisionalApplication Ser. No. 60/676,043 filed Apr. 29, 2005 and U.S. ProvisionalApplication Ser. No. 60/716,630 filed Sep. 13, 2005.

This invention relates to biological uses of nanoceria particles, and inparticular to methods and compositions useful for neuronal protection inretinal cells in vitro and the protection of mammalian cells fromreactive oxygen species in vivo and is supported in part by funding fromthe National Science Foundation and National Institutes of Health underthe Contract numbers: P20 RR17703, FY014427, FY13050, and FY12190.

FIELD OF THE INVENTION Background and Prior Art

Cerium is a silvery metallic element, belonging to the lanthanide group.Cerium Oxide (CeO₂) is used in precision polishing and lappingapplications. Ultra fine nano-size cerium oxide, less than 10nanometers, is more efficient for coating purposes. Recently, it wasreported by B. Rzigalinski et al. that nanoparticles prolong the life ofcortical neurons in culture 4 fold over the cells without treatment;decreased the intracellular Ca2+ concentration and prevented UV damageof cortical neurons. See B. Rzigalinski et al., “Cerium OxideNanoparticles Extend Cell Longevity and Act as Free Radical Scavengers”at website http://www.med.miami.edu/mnbws/Rzigalinski112.html. Based onits chemical characteristics, this effect is partially due to a decreaseof reactive oxygen species (ROS).

Retina tissue generates a large amount of ROS which are involved inlight-induced retina degeneration and age-related macular degeneration(AMD). The present invention tests the hypothesis that nanoparticles canpromote the lifespan of retinal neurons in culture and protect them fromapoptosis induced by hydrogen peroxide (H₂O₂) in vitro by decreasing theintracellular concentration of reactive oxygen species.

In 2004, T. H. Margrain et al. discuss the state of research in thetreatment of age-related macular degeneration in Progress in Retinal andEve Research, 2004, 23: 523-531, “Do Blue Light Filters ConferProtection Against Age-Related Macular Degeneration?” The problem ofapoptosis in the body is discussed in an article by P. Moongkarndi etal. in “Antiproliferation, Antioxidation and Induction of Apoptosis byGarcinia Mangostana (Mangosteen) on SKBR3 Human Breast Cancer CellLine”, Jl. of Ethnopharmacology, 2004, 90: 161-166.

Often persons suffering from light-induced retina degeneration andage-related macular degeneration (AMD) are without satisfactory remediesto prevent the eventual outcome of blindness. There are some proteinsavailable for neuronal protection of retinal cells, however, they arebig molecules and over time their effect may fade away.

It is desirable to find reliable solutions to prolong the lifespan ofretinal neurons so that blindness is avoided for persons with retinadegeneration and AMD.

In addition to diseases of the eye, many human diseases are due to thedeath of cells in specific tissues or organs. The majority of thosediseases are due to accumulation of metabolic insults from reactiveoxygen species originating within or outside of the cells. Thesediseases include all forms of blindness whether hereditary,light-induced, or physical damage such as occurs in retinal detachment.In addition, damage due to ageing, stroke, cardiac infarction, burns,etc, which proceed through reactive oxygen species, can be addressedwith the nanoceria particles of the present invention.

The present invention promotes a longer lifespan for retinal neurons.The greatest benefit of the nanoceria is its ability to get inside thecells and provide protection from reactive oxygen species (ROS); otherbody systems and tissues can also be protected from damage due to ROS.

SUMMARY OF THE INVENTION

A primary objective of the present invention is to promote the lifespanof retinal neurons in culture.

A secondary objective of the present invention is to protect retinalneurons from apoptosis induced by hydrogen peroxide (H₂O₂) in vitro.

A third objective of the present invention is to protect retinal neuronsfrom apoptosis induced by reactive oxygen species in vivo.

A fourth objective of the present invention is to inhibit the rise inthe intracellular concentration of reactive oxygen species (ROS).

A fifth objective of the present invention is to provide method forinhibiting apoptosis induced by H₂O₂ of retinal neurons in vitro in adose and time dependent manner.

A sixth objective of the present invention is to provide method forinhibiting apoptosis induced by reactive oxygen species in retinalneurons in vivo in a dose and time dependent manner.

A seventh objective of the present invention is to provide a method forpreventing an increase in the intracellular reactive oxygen species(ROS) in a dose and time dependent manner.

An eighth objective of the-present invention is to manufacture andmodify cerium oxide (CeO₂) nanoparticles for effective use in neuronalprotection in retinal cells.

A ninth objective of the present invention is to manufacture and modifycerium oxide (CeO₂) nanoparticles for effective use in mammalian cellsin vivo to inhibit damage caused by reactive oxygen species (ROS).

A preferred composition for promoting longevity of retinal neuronsincludes at least one of CeO.sub.n1 wherein 0<n1<2, and 0<n2<3 in theform of ultra-fine particles. The preferred ultra-fine particles have adiameter in a range between approximately 1 nanometer (nm) andapproximately 10 nm and the preferred CeO.sub.n1 is further defined asn1 equals approximately 2.

A preferred composition for inhibiting apoptosis induced by hydrogenperoxide oxidation of retinal neurons includes at least one ofCeO.sub.n1 wherein 0<n1<2, and 0<n2<3 in the form of ultra-fineparticles. The preferred ultra-fine particles have a diameter in a rangebetween approximately 1 nanometer (nm) and approximately 10 nM and thepreferred CeO.sub.n1 is further defined as n1 equals approximately 2.

A preferred composition for inhibiting apoptosis of retinal neurons in adose and time dependent manner includes at least one of CeO.sub.n1wherein 0<n1<2, and 0<n2<3 in the form of ultra-fine particles. Thepreferred ultra-fine particles have a diameter in a range betweenapproximately 1 nanometer (nm) and approximately 10 nm and the preferredCeO.sub.n1 is further defined as n1 equals approximately 2.

A more preferred composition that decreases the concentration ofintracellular reactive oxygen species (ROS) includes at least one ofCeO.sub.n1 wherein 0<n1<2, and 0<n2<3 in the form of ultra-fineparticles. The more preferred composition is used in the treatment ofdiseases of the retina selected from the group consisting oflight-induced retina degeneration and age-related macular degeneration,and is also used in vivo for the treatment of diseases in mammaliancells to inhibit damage caused by reactive oxygen species (ROS).

The mammalian cells that can be treated by the composition of thepresent invention, include, but are not limited to, retinal neurons,brain cells, heart cells, skin cells, liver cells, kidney cells andperipheral nervous system cells.

A preferred method for promoting longevity of retinal neurons includespreparing ultra-fine particles of at least one of CeO.sub.n1 wherein0<n1<2, and 0<n2<3 in a preselected concentration, and adding thepreselected concentration of CeO.sub.n1 wherein 0<n1<2, and 0<n2<3 toprimary retinal neurons. The preferred ultra-fine particles have adiameter in a range between approximately 1 nanometer (nm) andapproximately 10 nm and the CeO.sub.n1 is further defined as n1 equalsapproximately 2. The preferred preselected concentrations of CeO₂ are ina range between approximately 3 nanomolar (nM) and approximately fiftynanomolar (nM), more preferably in a range between approximately 3nanomolar (nM) and approximately twenty nanomolar (nM).

It is also preferred that the preselected concentrations of CeO₂ areadded to primary retinal neurons in vitro and/or administered tomammalian cells in vivo to protect the mammalian body system from damageto any tissue due to reactive oxygen species (ROS).

Further objects and advantages of the present invention will be apparentfrom the following detailed description of a presently preferredembodiment which is illustrated schematically in the accompanyingdrawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a timeline of exposure of a primary retinal neuron culture totreatment with cerium oxide nanoparticles.

FIG. 2 is a graph showing the percentage of apoptotic retinal neurons inculture with and without 5 nanomoles (nM) cerium oxide nanoparticletreatment at different time points.

FIG. 3 shows the initial cerium oxide nanoparticle treatments followedby hydrogen peroxide incubation, and time line of exposure.

FIG. 4A is a flow cytometry plot of the control sample of primaryretinal neurons with no cerium oxide nanoparticle treatments and nohydrogen peroxide incubation.

FIG. 4B is a flow cytometry plot of retinal neurons in the presence of100 micromoles (μM) hydrogen peroxide.

FIG. 4C is. a flow cytometry plot of retinal neurons treated with 1 nMcerium oxide in the presence of 100 micromoles (μM) hydrogen peroxide.

FIG. 4D is a flow cytometry plot of retinal neurons treated with 3 nMcerium oxide in the presence of 100 micromoles (μM) hydrogen peroxide.

FIG. 4E is a flow cytometry plot of retinal neurons treated with 5 nMcerium oxide in the presence of 100 micromoles (μM) hydrogen peroxide.

FIG. 4F is a flow cytometry plot of retinal neurons treated with 10 nMcerium oxide in the presence of 100 micromoles (μM) hydrogen peroxide.

FIG. 4G is a flow cytometry plot of retinal neurons treated with 20 nMcerium oxide in the presence of 100 micromoles (μM) hydrogen peroxide.

FIG. 4H is a flow cytometry plot of retinal neurons treated with 20 nMcerium oxide. FIG. 5 shows relative viable retinal neurons with andwithout incubation of different concentrations of cerium oxidenanoparticles for time periods between approximately 12 hours andapproximately 96 hours.

FIG. 6 shows the initial cerium oxide nanoparticle treatments, hydrogenperoxide and DCFH-DA incubation, and time line of exposure.

FIG. 7A is a graph of the intracellular level of reactive oxygen species(ROS) of retinal neurons after 30 minutes incubation with cerium oxidenanoparticles.

FIG. 7B is a graph of the intracellular level of reactive oxygen species(ROS) of retinal neurons after 12 hours incubation with cerium oxidenanoparticles.

FIG. 7C is a graph of the intracellular level of reactive oxygen species(ROS) of retinal neurons after 24 hours incubation with cerium oxidenanoparticles.

FIG. 7D is a graph of the intracellular level of reactive oxygen species(ROS) of retinal neurons after 96 hours incubation with cerium oxidenanoparticles.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Before explaining the disclosed embodiments of the present invention indetail it is to be understood that the invention is not limited in itsapplication to the details of the particular arrangements shown sincethe invention is capable of other embodiments. Also, the terminologyused herein is for the purpose of description and not of limitation.

The present invention has two embodiments describing in detail the invitro and in vivo treatment of mammalian cells with ultra fine nano-sizecerium oxide particles, less than 10 nanometers in diameter, to protectthe “body system” from damage to any tissue due to reactive oxygenspecies (ROS).

Hydrogen peroxide (H₂O₂) is one of many reactive oxygen species. In thepresent invention, H₂O₂ is added directly to cultures for the in vitrotreatments. H₂O₂ is not added to the live tissue samples, since H₂O₂ isone of the ROS products of light damage. The discussion below confirmsthat nanoceria particles inhibit all forms of reactive oxygen species(ROS).

For example, the nanoceria particles of the present invention canprotect the brain against stroke and reperfusion injury, the heart cellsfrom effects of cardiac infarction, the skin from UV rays and buminjuries. Neurodegeneration (e.g., Alzheimer's, Parkinson's, dementia,amyotrophic lateral sclerosis) and potentially mental retardation (dueto loss of brain cells) within the central and peripheral nervoussystems can also be inhibited. This protection can extend to diseaseswhich produce chronic problems such as cirrhosis of the liver or kidneyor the multi-organ effects of aging itself. The nanoceria can become theuniversal treatment for all major and minor diseases and events whichinvolve reactive oxygen species.

The nanoceria (CeO₂ nanoparticles) have the ability to destroy toxicproducts of metabolism known as reactive oxygen species (ROS). It hasbeen shown in one embodiment that the nanoceria particles prevent theROS induced death of mammalian retinal neurons in culture (in vitro) andsubsequently prolonged the life of the cells in culture and protectedthe cells from ROS. In the second embodiment, the ability to providemammalian cell protection in vivo is disclosed.

The first embodiment of the present invention provides a method andcomposition for promoting the lifespan of retinal neurons and protectingthe nerve cells in the eye from apoptosis induced by hydrogen peroxide(H₂O₂) in vitro by decreasing generation of intracellular reactiveoxygen species. The treatment of the eye with ultra fine nano-sizecerium oxide is a significant advance in biological uses of ceriumoxide. Persons afflicted with such conditions as, light-induced retinadegeneration and age-related macular degeneration (AMD) have hope forbrighter, clearer vision.

The examples below provide further detail on the preparation andtreatment of retinal nerve cells with CeO₂ nanoparticles.

EXAMPLE 1

A primary retinal neuron culture is obtained from albino rat pups.Retinae of Sprague-Dawley albino rat pups (0-2-day old) were dissectedout and mechanically dissociated in 25 ml of DMEM/F12 medium. Afterbeing filtered through 230 μm and 140 μm sieves, the dissociated cellswere centrifuged at 1200 rpm for 5 min. The cell pellets werere-suspended in the medium to 1×10⁵ cells/ml. 1 ml of the cellsuspension was plated in each well pre-treated with 10 μg/ml ofpoly-D-lysine. The cells were maintained in the medium until day 7, whendifferent concentrations of CeO₂ nanoparticles were added to thecultures. The timeline for the addition of CeO₂ nanoparticles is shownin FIG. 1. The treated neuronal cells were harvested on day 14, day 19,day 24 and day 29 after the beginning of treatment on day 7. Thepercentage of apoptotic retinal neurons in the culture with and without5 nM CeO₂ nanoparticle treatment is shown in FIG. 2 at the 12^(th) day,14^(th) day, 19^(th) day, 24^(th) day and 29^(th) day. Data are shown inM±S.D. Statistics were collected by Student t-test (n=3, *p<0.05,**p<0.01). FIG. 2 confirms that at every testing period the control withno CeO₂ nanoparticle treatment had a higher percentage of apoptoticretinal neurons in the culture, in contrast to the decreased percentageof apoptotic retinal neurons in the cells treated with 5 nM CeO₂nanoparticles.

EXAMPLE 2

The detection of apoptosis by flow cytometry is illustrated in FIGS. 3,4A-4H and 5. After periods of incubation with CeO₂ or H₂O₂, the cellswere washed with serum free medium 3 times, followed by treatment of 1ml of 1× trypsin for 2 min. After centrifuging, the cell pellet wasresuspended in 500 μl of 1×PBS containing 5 μl of Annexin V-FITC and 25μl of Propidium Iodide (PI). The kit used for the analysis iscommercially available from Beckman Coulter and is known as the “ANNEXINV-FITC Kit.” The mixture was incubated on ice for 10 minutes. Thefluorescent emissions of FITC and PI were detected by flow cytometry(Beckman Coulter) with the excitation filters of 492 nanometers (nm) and550 nm. The FITC fluorescent emissions signals the presence of cellsundergoing apoptosis; whereas, the PI signals with an automatic redcolor fluorescence the binding to DNA fragments identifying cells in anecrotic stage.

FIG. 3 shows a treatment timeline with CeO₂ incubation after the 7^(th)day of treatment at the time intervals of 12 hours, 24 hours, 72 hoursand 96 hours with each treated sample subsequently exposed to 12 hoursincubation time with H₂O₂.

FIGS. 4A-4H are representative flow cytometry plots of retinal neuronswith and without incubation with CeO₂ nanoparticles. Measurements weretaken after 96 hours. The cytometry plot shows activity of approximately10,000 cells in four quadrants, as described below.

C1 represents the percentage of 10,000 cells showing Annexin V positivesignals, which are interpreted to indicate the percentage of 10,000cells undergoing apoptosis.

C2 represents the percentage of 10,000 cells showing both Annexin V andPI positive signals, which is interpreted as the percentage of 10,000cells which are in late apoptotic or necrotic stage. (Annexin V signalsapoptotic ;stage; PI signals necrotic stage.)

C3 represents the percentage of 10,000 cells showing neither Annexin Vnor PI positive signals, which is interpreted as the percentage of10,000 cells which are still viable.

C4 represents the percentage of 10,000 cells showing a PI positivesignal, which is interpreted as the percentage of 10,000 cells in anecrotic stage.

Quadrants C2 and C4 show the percentage of 10,000 cells committed todie. In quadrant C1 the percentage of 10,000 cells undergoing apoptosisare shown and some may be salvaged. It is important to observe thepercentage of 10,000 cells in quadrant C2 for the efficacy of the ceriumoxide treatment of the present invention.

Focusing on the viable cells, the control in FIG. 4A has no CeO₂nanoparticle treatment and 73.9% of the cell population is viable after96 hours. FIG. 4B is treated with 100 μM H₂O₂, causing a deadly assaultand leaving the lowest percentage (57.7%) of viable cells. The additionof gradually increasing concentrations of cerium oxide with 100 μM H₂O₂,are shown in FIGS. 4C-4G.

FIG. 4C is treated with 1 nM CeO₂ nanoparticles and 100 μM H₂O₂ and60.6% of the cell population remains viable. FIG. 4D is treated with 3nM CeO₂ nanoparticles and 100 μM H₂O₂ and 67.0% of the cell populationis viable. FIG. 4E is treated with 5 nM CeO₂ nanoparticles and 100 μMH₂O₂, with 68.9% of the cell population remaining viable. FIG. 4F istreated with 10 nM CeO₂ nanoparticles and 100 μM H₂O₂ and 72.6% of thecell population is viable. FIG. 4G is treated with 20 nM CeO₂nanoparticles and 100 μM H₂O₂ and 72.3% of the cell population remainsviable.

FIG. 4H is treated with 20 nM CeO₂ nanoparticles without the addition ofH₂O₂ and 73.4% of the cell population remains viable showing that theCeO₂ nanoparticles alone have no negative effect on the cell population.

The data in FIGS. 4A-4H can also be summarized from the analysis ofcells undergoing apoptosis as shown in quadrant C1. The control, FIG. 4Ashows 16.2% of cells undergoing apoptosis under normal conditions,without any treatment, after 96 hours. FIG. 4B shows 26.1% of cellsundergoing apoptosis after the 100 μM H₂O₂ challenge. FIGS. 4C-4G showthere are 28.3%, 21.9%, 22.2%, 18.5% and 15.8% of cells undergoingapoptosis with 1, 3, 5, 10 and 20 nM CeO₂ nanoparticle treatment,respectively. FIG. 4H shows 15.7% of cells undergoing apoptosis with 20nM CeO₂ nanoparticle treatment, which is a slight improvement over notreatment at all as shown by the control in FIG. 4A.

Thus, cerium oxide nanoparticles inhibit apoptosis in retinal neurons invitro in a dose and time dependent manner.

FIG. 5 shows relative viable retinal neurons with and without incubationof different concentration of CeO₂ nanoparticles for different timeperiods. The concentration of CeO₂ nanoparticles were 1 nM, 3 nM, 5 nM,10 nM, 20 nM each with 100 μM H₂O₂; one sample was treated with only 100μM H₂O₂ and another sample was treated with only 20 nM CeO₂nanoparticles. The measurement of relative viable cells for each groupwas determined after 12 hours, 24 hours, 48 hours, 72 hours and 96hours. The effects showed dose and time dependency. 5 nM cerium oxidenanoparticles started to decrease the apoptosis at 24 h of incubation.As incubation time increased, the protective effect from 5 nMnanoparticles was more significantly increased. Additionally, 100 nM and20 nM nanoparticles began to have effects late with 96 h of incubation.(Statistical analysis was done by ANOVA, and Duncan test for post hocanalysis. Data are presented in M±S.D. n≧3, *p<0.05, **p<0.01). FIG. 5shows that among the various treatment doses, 5nM CeO₂ nanoparticletreatment gave the earliest response with moderate protective effect and20 nM CeO₂ nanoparticle treatment gave the highest protective effectwith late response.

EXAMPLE 3

Intracelluar reactive oxygen species (ROS) production was measured inboth CeO₂ nanoparticle treated and control cells using29,79-dichlorofluorescein diacetate (DCFH-DA, Sigma). Briefly, theretinal neurons were exposed to CeO₂ nanoparticles with differentconcentrations and various incubation times. After incubation, the cellswere incubated with 10 μM DCFH-DA (dissolved in dimethylsulfoxide(DMSO)) at 37° C. for 30 min. The cells then were incubated with 1 mMH₂O₂ at 37° C. for 30 min after the excess DCFH-DA was washed with PBS.The cells were harvested as described above. The intensity offluorescence was detected by flow cytometry with the excitation filterof 485 nm. The ROS level was calculated as a ratio: ROS=mean intensityof treated cells divided by mean intensity of control cells.

FIG. 6 is an experimental paradigm showing the beginning of the CeO₂nanoparticle treatment on the 7^(th) day and simultaneous incubationwith H₂O₂ and DCFH-DA and a timeline of exposure at 30 minutes, 12hours, 24 hours, and 96 hours. FIGS. 7A-7D show how the CeO₂nanoparticles decreased the generation of ROS in a dose and timedependent manner. The ROS generated in the groups of 5 nM, 10 nM, and 20nM nanoparticle incubation were statistically significantly less thanthe group without treatment at the earliest (12 h) of the tested points.3 nM nanoparticle incubation had an effect at 24 h. However, 1 nMnanoparticles did not show any significant decrease within the testedtime point. Statistical analysis was done by ANOVA, and Duncan test forpost hoc analysis. Data were shown in M±S.D. n=3,** p<0.01; *p<0.05.

The cerium oxide of the present invention includes those ceriumcompounds that have reacted with atmospheric oxygen to have stable oxidelayers identified as CeO.sub.n1, wherein O is less than 1 or equal to 2(0<n1<2) and (0<n2<3).

The cerium oxide particles of the present invention are characterized asultra-fine and are preferably in a size range of from approximately 1nanometer in diameter to approximately 10 nanometers in diameter; morepreferably from approximately 1 nm to approximately 7 nm. A judiciousselection of particle size is required by someone skilled in the art andis not a limitation of the present invention.

The results of testing in the above examples document the ability ofcerium oxide nanoparticles to promote the longevity of retinal neuronsin vitro and inhibit apoptosis induced by hydrogen peroxide on retinalneurons in vitro in a dose and time dependent manner. It has also beendetermined that cerium oxide nanoparticles decrease generation ofintracellular reactive oxygen species in a dose and time dependentmanner. Thus, the present invention represents a significant advance inthe treatment in degenerative diseases of the retina, such as, but notlimited to light-induced retina degeneration and age-related maculardegeneration (AMD).

In the second embodiment of the present invention a rat “light damage”model for retinal degeneration was used as the test system. The datademonstrate that the nanoceria prevented the death of retinal neuronswhen given prior to the “light insult”. The nanoceria particlesprotected the cells at the time of exposure as well as prevented thesubsequent death seen days later in the untreated animals. The in vivoroute of administration, including, direct injection into the eye,intravenous, intraperitoneal, intramuscular, oral or topically on theeye or skin may improve the result. Similarly, the time ofadministration of the nanoparticle, both before or after an insult, isimportant. Thus, the nanoceria will also prevent the death of retinalcells due to glaucoma, diabetic retinopathy, inherited retinaldegeneration (for example, Retinitis Pigmentosa), macular degeneration,retinal detachment or any disease or event which proceeds through theproduction of ROS. These particles should preserve and prolong visionwhen administered in vivo.

EXAMPLE 4

Rats were injected intravitreally with 2 microliters (μl) of nanoceria(concentrations from 0.1 to 1.0 micromolar) three days before they wereexposed for six hours to a bright light (2700 LUX) in a light box. Theanimals were returned to normal lighting for 3.5 days, then killed, theeyes enucleated, fixed, processed for paraffin embedding, sectioned, andeither stained with H & E or processed for immunocytochemistry.

The number of photoreceptors remaining was determined with the H&Esections by using a microscope connected to a digital camera to recordthe images and then measuring the thickness of the outer nuclear layerevery 240 microns from the optic nerve along both the superior and theinferior retina to the ora seratta. The data was then plotted as retinathickness versus the distance from the optic nerve head. The changes inother layers of retinal cells were recorded with images but notquantified. Cells actively undergoing apoptosis were also visualizedusing a commercially available “Apoptosis kit” and recording themicroscopic images with a fluorescence microscope.

The data demonstrate that the nanoceria at all concentrations testedprevented the immediate death of retinal cells shortly after exposure tolight as well as the ongoing death seen days later in the untreatedanimals. We therefore have demonstrated in vivo that these nanoceriaparticles can protect cells within the rat retina from light-inducedcell death.

Prior to the present invention, it was not known that nanoceriaparticles could be used in vivo for preventing blindness or death ofretinal cells, or for preventing the death in vivo of any other celltype in any disease. The chemical properties of CeO2 nanoparticlesenable the destruction of reactive oxygen species (ROS) produced bytoxins and/or products of oxygen metabolism within cells.

Thus, nanoceria can protect any cell type from ROS induced damages.Diseases which could possibly be prevented, cured or ameliorated wouldinclude hereditary blindness, macular degeneration, glaucoma, diabeticretinopathy, retinal detachment, and other blinding diseases whichinvolve ROS would be potentially solved. Similarly, in other cellswithin the central nervous system (CNS), neuronal death in strokes,degenerative diseases such as Alzheimer's Disease, Parkinson's,Huntington's Disease, and the death of peripheral nerves are preventableor at least the rate of cell death can be decreased and would result inprolonged function of the cells, tissues, organs, and individual.

While the invention has been described, disclosed, illustrated and shownin various terms of certain embodiments or modifications which it haspresumed in practice, the scope of the invention is not intended to be,nor should it be deemed to be, limited thereby and such othermodifications or embodiments as may be suggested by the teachings hereinare particularly reserved especially as they fall within the breadth andscope of the claims here appended.

1-13. (canceled)
 14. A method for promoting longevity of retinal neuronscomprising the steps of: preparing ultra-fine particles of at least oneof CeO.sub.n1 wherein 0<n1<2, and 0<n2<3 in a preselected concentration;and adding the preselected concentration of CeO.sub.n1 wherein 0<n1<2,and 0<n2<3 to primary retinal neurons.
 15. The method of claim 14,wherein the ultra-fine particles have a diameter in a range betweenapproximately 1 nanometer (nm) and approximately 10 nm.
 16. The methodof claim 14, wherein the CeO.sub.n1 is further defined as n1 equalsapproximately
 2. 17. The method of claim 16, wherein the preselectedconcentrations of CeO₂ are in a range between approximately 3 nanomolar(nM) and approximately fifty nanomolar (nM).
 18. The method of claim 17,wherein the preselected concentrations of CeO₂ are in a range betweenapproximately 3 nanomolar (nM) and approximately twenty nanomolar (nM).19. The method of claim 14, wherein the preselected concentrations ofCeO₂ are added to primary retinal neurons in vitro.
 20. The method ofclaim 14, wherein the preselected concentrations of CeO₂ areadministered to mammalian cells in vivo to protect the mammalian bodysystem from damage to any tissue due to reactive oxygen species (ROS).